Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

Abstract

BackgroundPhotosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.ResultsWe adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.ConclusionsOur results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

“…In pIY107, the C-terminus of HynS is modified with a strep-tag [8,30,31] and the N-terminus of HynL is modified with a His 6 -tag. Details of the construction of pIY107 will be published elsewhere, but we provide its sequence in the Additional file 1.…”

“…The upstream amplicon was generated by primers BC000AvrF and BC001AvrR; the downstream amplicon was generated by primers BC002AvrF and IY171HynSR [8]; sequences and descriptions of primers are provided in Additional file 1: Table S4. A three-piece Gibson isothermal assembly was then performed using both amplicons and pIY107 doubly digested with AgeI and AvrII.…”

“…A second gel containing 3 × replicates of tandem samples was subjected to western blot as described previously [8]. One set of replicates was curved on the gel, complicating the densitometry boxing procedure, so it was removed from analysis, although qualitatively it presented similar results.…”

Biotechnological hydrogen production by photosynthesis

“…The wild‐type enzyme favors hydrogen uptake activity over hydrogen evolution. However, a mutant with two substitutions in the Fe‐S cluster exhibited four times greater hydrogen evolution activity than the wild type .…”

Hydrogen production using photobiological methods

“…Liang et al (2009) knocked out membrane-bound [NiFe] hydrogenase (MBH) in photosynthetic bacterium Allochromatium vinosum to improve the H 2 production rate. Similarly, H 2 evolution activities were increased by the mutation of hupW, which encodes putative hydrogenase-specific protease in Nostoc strain PCC 7120 of cyanobacterium (Lindberg, Devine, Stensj€ o, & Lindblad, 2011), inactivation of functional uptake hydrogenase (hupS) in nitrogen-fixing cyanobacterium Anabaena siamensis TISTR 8012 (Khetkorn et al, 2012), and substitution of amino acid, which alters the ligation of Fe-S clusters of [NiFe] uptake hydrogenase even in marine bacterium Alteromonas macleodii (Yonemoto, Matteri, Nguyen, Smith, & Weyman, 2013).…”